We have constructed a putative full length cDNA clone of the genome that has been evaluated in both cell culture and by direct transfection of RNA transcripts of this clone into chimpanzee liver. While the original clone was not infectious, Rice has recently prepared new clones in which additional sequence has been added. These constructs are presently being evaluated. In vitro cell culture experiments continue using primarily a liver cell line, THLE5b. We have demonstrated the low level replication of HCV by quantitative PCR through 6 passages but have failed to show any increasing levels of replication and in fact have lost the virus at that passage level. These experiments are being repeated. In order to improve the replication in cell culture we are expressing various HCV non-structural proteins such as the polymerase in the cell cultures in the hopes that over expression may improve replication. As hepatocellular carcinoma is the most serious complication of chronic HCV infection, we have initiated experiments to try to understand the relationship of the virus to carcinogenesis. We have studied binding of all the individual HCV specific proteins to p53 and RB tumor suppressor antigens. These experiments were well controlled but showed no specific binding nor have we demonstrated cleavage of p53 and RB by the HCV proteases. We are also looking for hepatocellular carcinoma (HCC) specific tumor antigens as we have found that 3 out of four patients with HCC have antibodies directed at the surface of hepatoma derived cell lines. As we are also interested in CTL responses to HCC, we HLA typed 7 HCC derived cell lines but found that 6 had had down regulated HLA class I and only one allele of the HLA A-locus was expressed. We are isolating CTL directly from HCC tissue and testing them on HLA compatible HCC cell lines as targets. Established CTL lines will be used to identify specific genes coding for the HCC antigen.